To address the specific mission interests of the NIAAA: "alcohol-related immune dysregulation in HIV with and without coinfection and other comorbidities" and NIDA basic science research: effects of drug use on immune correlates in HIV and co-infection, this proposal will evaluate alcohol and opioid affects on innate immune cell phenotype and function in the context of HCV, HIV, and HCV-HIV disease. SPECIFIC AIM 1: To determine the effect of chronic alcohol ingestion and opioid use on MDC and PDC numbers and function in HCV, HIV, and HCV/HIV infected hosts. METHODS: Non-expanded, immature MDC and PDC will be purified from HCV, HIV, and HCV-HIV infected hosts who are identified with either minimal to moderate, or heavy alcohol consumption, and identified as either receiving, or not receiving methadone. Healthy control samples without chronic alcohol ingestion or opioid use will be used as a reference. Cells will be analyzed for levels of costimulatory molecule expression, antigen uptake ability, and maturation in response to TLR ligand (flow cytometry and ELISA). In parallel, freshly purified and TLR stimulated MDC and PDC will be analyzed for ability to activate allogeneic healthy control naive CD4 T cells or autologous NK cells (cytokine ELISPOT). Clinical parameters will be used to evaluate associations between cellular defects and HCV or HIV disease activity/stage. SPECIFIC AIM 2: To determine the direct effect of in vitro alcohol and opioid exposure on healthy control, HCV, HIV and HCV/HIV MDC and PDC function. METHODS: Aim 2 methodology will be identical to that used in Aim 1 with the following exceptions (1)MDC and PDC will be isolated only from HCV, HIV, HCV/HIV infected and healthy control subjects who are not ingesting heavy amounts of alcohol or opioids, and (2)MDC and PDC will be pre-treated in vitro with alcohol and/or morphine, and washed prior to assay start. RATIONALE: There is a high prevalence of alcohol and opioid use within the HCV, HIV and HCV-HIV populations. Heavy alcohol consumption has been associated with enhanced liver disease in HCV infected individuals and increased disease progression in HIV infected individuals. Moreover, opioid use has been associated with increased HIV disease progression and opioid exposure with suppression of IFN-a mediated anti-HCV activity. Understanding the innate cellular immune modulations caused by both substance abuse in chronically infected individuals and/or acute alcohol or opioid exposure may provide insight into mechanisms of disease pathogenesis and facilitate proper treatment designs.